Endothelial cells are contnuously subjected to fluid shear stress, and laminar shear stress regulates endothelial cell morphology and function by producing autocrine and paracrine factors. Shear stress has been shown to be an anti-atherogenic mechanical force and to protect endothelial cells from cell death. However, it is not known: 1) how endothelial cells sense the changes in shear stress; and 2) how this "mechanosensing" leads to specific regulation of early signaling events that control endothelial cell structure and function. Answering these questions is the long-term goal of this research program and will help to elucidate the pathogenic mechanisms underlying the focal development of atherosclerotic plaques and provide new molecular targets for therapeutic intervention. Understanding how cells respond to the changes in environment such as physical forces provides a critical perspective in cell biology and in pathophysiology. An important molecule involved in these cellular responses is a member of MAP kinase family, extracellular signal-regulated kinase (ERK). ERK is a signaling mediator linking extracellular stimuli to intracellular responses including cell growth, proliferation, differentiation and metabolism. Because of its critical role as a signaling mediator of transcription factors and subsequent gene expression, we have focused on characterizing the early mechano-signal transduction mechanisms regulating the ERK pathway. ERK is rapidly activated by shear stress in cultured bovine aortic endothelial cells (BAEC) in a shear force-dependent manner by mechanisms involving Gi2a, PKCe, Src, FAK, and Ras. However, the mechanisms by which endothelial cells control the signaling fidelity to activating ERK rapidly in response to shear stress is not known. In addition, the activation sequence of these signaling molecules have not been determined. The objective of this application is to define the spatial and temporal mechanisms maintaining the rapid and specific activation of ERK in response to shear stress. This proposal builds upon our recent evidence suggesting that this shear stress-dependent activation of ERK occurs in a signaling platform in the plasma membrane, called caveolae or caveolae-like domains. It is hypothesized that shear stress activates ERK rapidly and specifically by mechanisms dependent upon caveolin and caveolae-like domains, which provide a mechano- sensitive platform for the spatio-temporal organization of signaling components in endothelial cells. This hypothesis will be examined in BAEC through the pursuit of the following two specific aims. Aim I. Define the role of caveolin in shear- dependent activation of ERK. Aim II. Determine the role of caveolae in shear-dependent activation of ERK.